Multicellular organisms exert precise control over cell number. The normal structure and function of tissues depends on the maintenance of appropriate cell numbers. A balance between cell proliferation and cell death (White, E. (1996) Genes Dev. 10:1–15) achieves this homeostasis.
Cell death occurs in nearly every type of vertebrate cell via necrosis or through a suicidal form of cell death, known as apoptosis. Apoptosis is triggered by a variety of extracellular and intracellular signals that engage a common, genetically programmed death mechanism (Wyllie, A. H. (1995) Curr. Opin. Gen. Dev. 5:97–104). As a regulatory process, apoptosis influences diverse biological phenomena including the development of neural architecture, the immune system's ability to discriminate between self and non-self, and the expunging of redundant, damaged or infected cells.
It has become evident that many diseases are associated with dysregulation of the process of cell death. Experimental models have established a cause-effect relationship between derangement in the mechanism regulating apoptosis or necrosis and the pathenogenicity of various neoplastic, autoimmune and viral diseases (Thompson, C. B. (1995) Science 267:1456–1462). A well-defined example is the effect of aberrant, high-level expression of bcl-2 on lymphoma development. The bcl-2 oncogene was originally identified as the genetic element located at the t(14:18) chromosomal translocation breakpoint present in many B-cell follicular lymphomas (Korsmeyer, S. J. (1992) Blood 359:554–556). Since that discovery, it has been convincingly established that the bcl-2 gene product inhibits apoptosis induced by a variety of stimuli and that its oncogenic potential stems from its ability to derail apoptosis (Sentman, C. L. et al. (1994) Cell 67:878–888; and McDonnell, T. J. and Korsmeyer, S. J. (1991) Nature 349:254–256).
Failed or reduced apoptosis is associated with the development of human autoimmune lymphoproliferative syndrome as well as mouse models of this disease. MRL-lpr or gld mice develop lymphadenopathy, splenomegaly, nephritis and arthritis; as well they produce large quantities of autoantibodies (Cohen, P. L. and Eisenberg, R. A. (1991) Annu. Rev. Immunol. 9:243–269).
These mice carry loss of function mutations in the genes encoding FAS and Fas ligand, respectively (Adachi, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:1756–1760); Takakashi, T. et al. (1994) Cell 76:969–976). FAS, a ubiquitously expressed cell surface receptor, normally generates an apoptotic response upon binding with Fas ligand (Itoh, N. et al. (1991) Cell 66:233–243). In mice carrying these loss of function mutations, the disruption of FAS signaling renders T cells resistant to peripheral deletion by apoptosis (Russell, H. et al. Proc. Natl. Acad. Sci. USA 90:4409–4413). The inappropriate survival of these cells results in a pathologic accumulation of T and B cells evidenced by the neoplastic-like growth of lymphoid tissues and high-level autoantibody production. In humans, autoimmune lymphoproliferative syndrome shares similarities with the mouse phenotype including lymphadenopathy, splenomegaly, autoantibodies and autoimmune manifestations. Patients with this disease likewise carry mutation in the FAS gene (Nagata, S. (1998) J. Hum. Genet. 43:2–8).
Benzodiazepine compounds have been traditionally known to bind to benzodiazepine receptors in the central nervous system (CNS) and thus have been used to treat various CNS disorders including anxiety and epilepsy. More recently, peripheral benzodiazepine receptors have also been identified, which receptors may incidentally also be present in the CNS. Benzodiazepines and related structures have pro-apoptotic and cytotoxic properties useful in the treatment of transformed cells grown in tissue culture. There is therapeutic potential for this class of agents against cancer and other neoplastic diseases. Two specific examples shown are neuroblastoma and ovarian cancer.
Neuroblastoma is the most common extracranial solid tumor found in children. Modern treatments, which include chemotherapy, radiation therapy and surgery, have not significantly reduced the mortality of metastatic neuroblastoma. Novel therapies are needed to improve survival of children with this disease. We show that benzodiazepine compounds are able to slow the growth of these tumor cells. See, Sugimoto, T. et al. (1984) J. Natl. Cancer Inst. 73:51–57; Schwab, M. et al. (Sep. 15, 1983) Nature 305:245–248; and Dive, C. and Wyllie, A. H. (1993) Apoptosis and Cancer Chemotherapy, Oxford Blackwell, pp. 21–56.
Ovarian cancer is difficult to treat due to chemoresistance shown by the patient to standard chemotherapy drugs. Treatment failures are usually attributed to the emergence of chemotherapy resistant cells. We show that benzdiazepine compounds are able to kill ovarian cancer cells that are chemoresistant. See, Pestell, K. E. et al. (1998)Int. J. Cancer 77(6):913–918; Beale, P. J. et al. (2000) Br. J. Cancer 82(2):436–440; Ozols, R. F. (February 1999) Semin. Oncol. 26(1 Suppl. 2):84–89; Liu, J. R. et al. (September 1998) Gynecol. Oncol. 70(3):398–403; Chumakov, A. M. et al. (November 1993) Oncogene 8(11):3005–3011; and Raynaud, F. I. et al. (August 1996) Br. J. Cancer 74(3):380–386.
Several benzodiazepine analogs have been reported as analgesic and anti-inflammatory agents. See, for example, U.S. Pat. Nos. 4,076,823, 4,110,337, 4,495,101, 4,751,223 and 5,776,946.
U.S. Pat. Nos. 5,324,726 and 5,597,915 disclose that some benzodiazepines are antagonists of cholecystokinin and gastrin and thus might be useful to treat certain gastrointestinal disorders.
Certain benzodiazepines have also been explored as inhibitors of human neutrophil elastase and thus potentially useful to treat the human neutrophil elastase-mediated conditions such as myocardial ischemia, septic shock syndrome, among others. See U.S. Pat. No. 5,861,380.
U.S. Pat. No. 5,041,438 reported that certain benzodiazepines could be useful as anti-retroviral agents. However, as it will become apparent from the description below, the present invention provides novel methods and compositions that are distinct from the above-disclosed methods and compositions.